Fluorescent nucleotides are synthetic building blocks of DNA and RNA that, in addition to being artificial structural components, emit colored light when irradiated with light of a longer wavelength. Because the character of this emission is sensitive to the chemical environment, fluorescent nucleotides can be used as exquisitely sensitive visual probes of the activity of DNA-processing enzymes. A special emerging class of synthetic fluorescent nucleotides, tricyclic cytidine analogues, has the unique properties of maintaining fluorescence in double-stranded DNA (the double helix), being highly compatible with Nature's machinery for DNA and RNA synthesis (DNA and RNA polymerases), and minimally perturbing the nucleic acids, once incorporated. They are limited, however, in that they are available with colors in only a small range of the visible spectrum and they are an order of magnitude less bright than many common commercial fluors. In this project, new fluorescent nucleotides will be developed to overcome these limitations by systematically modifying the chemical structure of the lead compounds. Preliminary results indicate that the fluorescence wavelengths and intensities are strongly sensitive to substituent and other structural modifications to the tricyclic cytidine analogues. Using chemical synthesis, a new set of modifications will be systematically examined and a number of novel tri- and tetracyclic cytidine analogues frameworks will be prepared. The results of systematic substituent testing will be used iteratively to refine the strategic development of bright nucleotide fluors that offer absorption and emission wavelengths across a broader window of the visible spectrum. Concurrently with these fluorescence measurements, the new nucleotides will be used as test substrates for DNA and RNA polymerases, the results of which will be used to establish protocols for their incorporation into nucleic acids and to provide new knowledge of the mechanisms used by polymerases to discriminate between correct and incorrect incoming nucleotides. With the successful expansion of the absorption and emission window and the brightening of the dyes, new FRET (Fluorescence Resonance Energy Transfer) applications and direct in vivo fluorescence imaging of DNA synthesis are likely to be possible. Nucleic acid metabolism is an extremely complex and medically important process. For example, most cancer and antiviral therapeutics attack some aspect of this process. The proposed fluorescent nucleotides are expected to offer new methods for analyzing nucleic acid metabolism, which can lead to the development of novel therapeutics and new ways of using existing therapeutics, in addition to fundamental insights into the mechanisms of how cells metabolize nucleic acids. PUBLIC HEALTH RELEVANCE: DNA replication plays multiple critical roles in health care and disease - cellular and viral DNA replication are primary targets for cancer chemotherapeutics and antiviral agents, and incorrect replication can lead to multiple disease states, including cancer. The proposed studies will provide tools to better understand this process, which may then lead to more effective therapeutic agents.